From a recombinant key antigen to an accurate, affordable serological test: Lessons learnt from COVID-19 for future pandemics.

Serological tests detect antibodies generated by infection or vaccination, and are indispensable tools along different phases of a pandemic, from early monitoring of pathogen spread up to seroepidemiological studies supporting immunization policies. This work discusses the development of an accurate and affordable COVID-19 antibody test, from production of a recombinant protein antigen up to test validation and economic analysis. We first developed a cost-effective, scalable technology to produce SARS-COV-2 spike protein and then used this antigen to develop an enzyme-linked immunosorbent assay (ELISA). A receiver operator characteristic (ROC) analysis allowed optimizing the cut-off and confirmed the high accuracy of the test: 98.6% specificity and 95% sensitivity for 11+ days after symptoms onset. We further showed that dried blood spots collected by finger pricking on simple test strips could replace conventional plasma/serum samples. A cost estimate was performed and revealed a final retail price in the range of one US dollar, reflecting the low cost of the ELISA test platform and the elimination of the need for venous blood sampling and refrigerated sample handling in clinical laboratories. The presented workflow can be completed in 4 months from first antigen expression to final test validation. It can be applied to other pathogens and in future pandemics, facilitating reliable and affordable seroepidemiological surveillance also in remote areas and in low-income countries.

p53 reactivation with induction of massive apoptosis-1 (PRIMA-1) inhibits amyloid aggregation of mutant p53 in cancer cells.

p53 mutants can form amyloid-like structures that accumulate in cells. p53 reactivation with induction of massive apoptosis-1 (PRIMA-1) and its primary active metabolite, 2-methylene-3-quinuclidinone (MQ), can restore unfolded p53 mutants to a native conformation that induces apoptosis and activates several p53 target genes. However, whether PRIMA-1 can clear p53 aggregates is unclear. In this study, we investigated whether PRIMA-1 can restore aggregated mutant p53 to a native form. We observed that the p53 mutant protein is more sensitive to both PRIMA-1 and MQ aggregation inhibition than WT p53. The results of anti-amyloid oligomer antibody assays revealed that PRIMA-1 reverses mutant p53 aggregate accumulation in cancer cells. Size-exclusion chromatography of the lysates from mutant p53-containing breast cancer and ovarian cell lines confirmed that PRIMA-1 substantially decreases p53 aggregates. We also show that MDA-MB-231 cell lysates can "seed" aggregation of the central core domain of recombinant WT p53, corroborating the prion-like behavior of mutant p53. We also noted that this aggregation effect was inhibited by MQ and PRIMA-1. This study provides the first demonstration that PRIMA-1 can rescue amyloid-state p53 mutants, a strategy that could be further explored as a cancer treatment.

Functional Analysis and Fine Mapping of the 9p22.2 Ovarian Cancer Susceptibility Locus.

Genome-wide association studies have identified 40 ovarian cancer risk loci. However, the mechanisms underlying these associations remain elusive. In this study, we conducted a two-pronged approach to identify candidate causal SNPs and assess underlying biological mechanisms at chromosome 9p22.2, the first and most statistically significant associated locus for ovarian cancer susceptibility. Three transcriptional regulatory elements with allele-specific effects and a scaffold/matrix attachment region were characterized and, through physical DNA interactions, BNC2 was established as the most likely target gene. We determined the consensus binding sequence for BNC2 in vitro, verified its enrichment in BNC2 ChIP-seq regions, and validated a set of its downstream target genes. Fine-mapping by dense regional genotyping in over 15,000 ovarian cancer cases and 30,000 controls identified SNPs in the scaffold/matrix attachment region as among the most likely causal variants. This study reveals a comprehensive regulatory landscape at 9p22.2 and proposes a likely mechanism of susceptibility to ovarian cancer. SIGNIFICANCE: Mapping the 9p22.2 ovarian cancer risk locus identifies BNC2 as an ovarian cancer risk gene.See related commentary by Choi and Brown, p. 439.

Tissue factor mediates microvesicles shedding from MDA-MB-231 breast cancer cells.

Extracellular vesicles, such as microvesicles (MVs), were identified as important players in tumor progression and acquisition of an aggressive phenotype. Tissue factor (TF) is a transmembrane protein that initiates the blood coagulation cascade. In tumor cells, TF has been associated with aggressiveness and cancer progression. Previous studies demonstrate that TF is incorporated into MVs secreted by tumor cells; however, it is unknown whether TF is actively involved in the release of MVs. Here, we investigated the influence of TF expression on the release of MVs. TF silencing was achieved through CRISPR/Cas9 approaches in the human breast cancer cell line, MDA-MB-231. TF knockout in MDA-MB-231 cells efficiently reduced TF-dependent signaling and procoagulant activity. Remarkably, silencing of TF caused a significant decrease in the number of MVs released by MDA-MB-231 cells. We also observed an increase in actin-positive membrane projections in TF knockout cells and a reduction in RhoA expression when compared to TF-expressing cells. Treatment of MDA-MB-231 cells with the RhoA-ROCK signaling pathway inhibitor, fasudil, significantly reduced the release of MVs. Taken together, our results suggest a novel and relevant role for TF in tumor biology by playing an active role in the MVs secretion.

BRCA1 recruitment to damaged DNA sites is dependent on CDK9.

Double strand break lesions, the most toxic type of DNA damage, are repaired primarily through 2 distinct pathways: homology-directed recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1, 2 proteins containing the BRCT modular domain, play an important role in DNA damage response (DDR) by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified a putative interaction between BRCA1 and BARD1 (BRCA1-associated RING domain 1) and the cyclin-dependent kinase (CDK9). CDK9 is a component of the positive transcription elongation complex and has been implicated in genome integrity maintenance associated with the replication stress response. Here we show that CDK9 interacts with endogenous BRCA1 and BARD1 mediated by their RING finger and BRCT domains, and describe CDK9 ionizing radiation-induced foci (IRIF) formation and its co-localization with BRCA1 in DNA damage sites. Cells lacking CDK9 are characterized by an altered γ-H2AX foci dynamics after DNA damage, a reduced efficiency in HR but not in NHEJ repair, failure to form BRCA1 and RAD51 IRIF and increased sensitivity to genotoxic agents. These data indicate that CDK9 is a player in the DDR and is consistent with its participation in HR pathway by modulating BRCA1 response.

Enhancer scanning to locate regulatory regions in genomic loci.

This protocol provides a rapid, streamlined and scalable strategy to systematically scan genomic regions for the presence of transcriptional regulatory regions that are active in a specific cell type. It creates genomic tiles spanning a region of interest that are subsequently cloned by recombination into a luciferase reporter vector containing the simian virus 40 promoter. Tiling clones are transfected into specific cell types to test for the presence of transcriptional regulatory regions. The protocol includes testing of different single-nucleotide polymorphism (SNP) alleles to determine their effect on regulatory activity. This procedure provides a systematic framework for identifying candidate functional SNPs within a locus during functional analysis of genome-wide association studies. This protocol adapts and combines previous well-established molecular biology methods to provide a streamlined strategy, based on automated primer design and recombinational cloning, allowing one to rapidly go from a genomic locus to a set of candidate functional SNPs in 8 weeks.

Phosphatidylinositol-3-kinase as a putative target for anticancer action of clotrimazole.

Clotrimazole (CTZ) has been proposed as an antitumoral agent because of its properties that inhibit glycolytic enzymes and detach them from the cytoskeleton. However, the broad effects of the drug, e.g., acting on different enzymes and pathways, indicate that CTZ might also affect several signaling pathways. In this study, we show that CTZ interferes with the human breast cancer cell line MCF-7 after a short incubation period (4 h), thereby diminishing cell viability, promoting apoptosis, depolarizing mitochondria, inhibiting key glycolytic regulatory enzymes, decreasing the intracellular ATP content, and permeating plasma membranes. CTZ treatment also interferes with autophagy. Moreover, when the incubation is performed under hypoxic conditions, certain effects of CTZ are enhanced, such as phosphatidylinositol-3-phosphate kinase (PI3K), which is inhibited upon CTZ treatment; this inhibition is potentiated under hypoxia. CTZ-induced PI3K inhibition is not caused by upstream effects of CTZ because the drug does not affect the interaction of the PI3K regulatory subunit and the insulin receptor substrate (IRS)-1. Additionally, CTZ directly inhibits human purified PI3K in a dose-dependent and reversible manner. Pharmacologic and in silico results suggest that CTZ may bind to the PI3K catalytic site. Therefore, we conclude that PI3K is a novel, putative target for the antitumoral effects of CTZ, interfering with autophagy, apoptosis, cell division and viability.

BRCA1 Circos: a visualisation resource for functional analysis of missense variants.

BACKGROUND: Inactivating germline mutations in the tumour suppressor gene BRCA1 are associated with a significantly increased risk of developing breast and ovarian cancer. A large number (>1500) of unique BRCA1 variants have been identified in the population and can be classified as pathogenic, non-pathogenic or as variants of unknown significance (VUS). Many VUS are rare missense variants leading to single amino acid changes. Their impact on protein function cannot be directly inferred from sequence information, precluding assessment of their pathogenicity. Thus, functional assays are critical to assess the impact of these VUS on protein activity. BRCA1 is a multifunctional protein and different assays have been used to assess the impact of variants on different biochemical activities and biological processes. METHODS AND RESULTS: To facilitate VUS analysis, we have developed a visualisation resource that compiles and displays functional data on all documented BRCA1 missense variants. BRCA1 Circos is a web-based visualisation tool based on the freely available Circos software package. The BRCA1 Circos web tool (http://research.nhgri.nih.gov/bic/circos/) aggregates data from all published BRCA1 missense variants for functional studies, harmonises their results and presents various functionalities to search and interpret individual-level functional information for each BRCA1 missense variant. CONCLUSIONS: This research visualisation tool will serve as a quick one-stop publically available reference for all the BRCA1 missense variants that have been functionally assessed. It will facilitate meta-analysis of functional data and improve assessment of pathogenicity of VUS.

Probing structure-function relationships in missense variants in the carboxy-terminal region of BRCA1.

Germline inactivating variants in BRCA1 lead to a significantly increased risk of breast and ovarian cancers in carriers. While the functional effect of many variants can be inferred from the DNA sequence, determining the effect of missense variants present a significant challenge. A series of biochemical and cell biological assays have been successfully used to explore the impact of these variants on the function of BRCA1, which contribute to assessing their likelihood of pathogenicity. It has been determined that variants that co-localize with structural or functional motifs are more likely to disrupt the stability and function of BRCA1. Here we assess the functional impact of 37 variants chosen to probe the functional impact of variants in phosphorylation sites and in the BRCT domains. In addition, we perform a meta-analysis of 170 unique variants tested by the transcription activation assays in the carboxy-terminal domain of BRCA1 using a recently developed computation model to provide assessment for functional impact and their likelihood of pathogenicity.

Characterization of LGALS3 (galectin-3) as a player in DNA damage response.

DNA damage repair (DDR) is an orchestrated process encompassing the injury detection to its complete resolution. DNA double-strand break lesions are repaired mainly by two distinct mechanisms: the error-free homologous recombination (HR) and the error-prone non-homologous end-joining. Galectin-3 (GAL3) is the unique member of the chimeric galectins subfamily and is reported to be involved in several cancer development and progression related events. Recently our group described a putative protein interaction between GAL3 and BARD1, the main partner of breast and ovarian cancer susceptibility gene product BRCA1, both involved in HR pathway. In this report we characterized GAL3/BARD1 protein interaction and evaluated the role of GAL3 in DDR pathways using GAL3 silenced human cells exposed to different DNA damage agents. In the absence of GAL3 we observed a delayed DDR response activation, as well as a decrease in the G 2/M cell cycle checkpoint arrest associated with HR pathway. Moreover, using a TAP-MS approach we also determined the protein interaction network of GAL3.

GWAS meta-analysis and replication identifies three new susceptibility loci for ovarian cancer.

Genome-wide association studies (GWAS) have identified four susceptibility loci for epithelial ovarian cancer (EOC), with another two suggestive loci reaching near genome-wide significance. We pooled data from a GWAS conducted in North America with another GWAS from the UK. We selected the top 24,551 SNPs for inclusion on the iCOGS custom genotyping array. We performed follow-up genotyping in 18,174 individuals with EOC (cases) and 26,134 controls from 43 studies from the Ovarian Cancer Association Consortium. We validated the two loci at 3q25 and 17q21 that were previously found to have associations close to genome-wide significance and identified three loci newly associated with risk: two loci associated with all EOC subtypes at 8q21 (rs11782652, P = 5.5 × 10(-9)) and 10p12 (rs1243180, P = 1.8 × 10(-8)) and another locus specific to the serous subtype at 17q12 (rs757210, P = 8.1 × 10(-10)). An integrated molecular analysis of genes and regulatory regions at these loci provided evidence for functional mechanisms underlying susceptibility and implicated CHMP4C in the pathogenesis of ovarian cancer.

Ketoprofen impairs immunosuppression induced by severe sepsis and reveals an important role for prostaglandin E2.

The mechanism of immunosuppression induced by severe sepsis is not fully understood. The production of prostaglandin E2 (PGE2) during sepsis is well known, but its role in long-term consequences of sepsis has not been explored. The current study evaluates the role of PGE2 in the development of immunosuppression secondary to sepsis and its potential as therapeutic target. Cecal ligation and puncture was used as an experimental model for sepsis induction in Balb/c and C57BL/6 mice. Immunosuppression was evaluated by the response to secondary infection with Aspergillus fumigatus in sepsis survivors. The role of prostanoids was evaluated in vivo and in vitro by treatment with the cyclooxygenase inhibitor ketoprofen. Balb/c mice were more susceptible than C57BL/6 to severe sepsis and to secondary infection, with a greater mortality rate. Prostaglandin E2 concentrations found in bronchoalveolar lavage in sham and cecal ligation and puncture group after fungal challenge were much higher in Balb/c than in C57BL/6 mice. Ketoprofen treatment improved survival of septic Balb/c mice subjected to secondary infection, while also enhancing macrophage phagocytosis and neutrophil recruitment to the lungs. We identified a pivotal role for PGE2 acting on EP4 receptors in modulating cytokine production differentially by sham and septic macrophages. Furthermore, sepsis also altered key enzymes in PGE2 synthesis and degradation. Our results indicate the involvement of PGE2 in severe sepsis-induced immunosuppression. Inhibition of PGE2 production represents an attractive target to improve innate immune response against secondary infection in the immunocompromised host.

Modulation of expression and activity of cytochrome P450s and alteration of praziquantel kinetics during murine schistosomiasis.

In this study, we investigated the expression and activity of liver cytochrome P450s (CYPs) and praziquantel (PZQ) kinetics in mice infected with Schistosoma mansoni. Swiss Webster (SW) mice of both genders were infected (100 cercariae) on postnatal day 10 and killed on post-infection days (PIDs) 30 or 55. Non-infected mice of the same age and sex served as controls. Regardless of mouse sex, infection depressed the activities of CYP1A [ethoxy/methoxy-resorufin-O-dealkylases (EROD/MROD)], 2B9/10 [pentoxy/benzyloxy-resorufin-O-dealkylases (PROD, BROD)], 2E1 [p-nitrophenol-hydroxylase (PNPH)] and 3A11 [erythromycin N-demethylase (END)] on PID 55 but not on PID 30. On PID 55, infection decreased liver CYP mRNA levels (real-time reverse transcription-polymerase chain reaction). On PID 30, whereas mRNA levels remained unaltered in males, they were depressed in females. Plasma PZQ (200 and 400 mg/kg body weight intraperitoneally) levels were measured (high-performance liquid chromatography) at different post-treatment intervals. In males and females, infection delayed the PZQ clearance on PID 55, but not on PID 30. Therefore, it can be concluded that schistosomiasis down-modulated CYP expression and activity and delayed PZQ clearance on PID 55, when a great number of parasite eggs were lodged in the liver. On PID 30, when egg-laying was initiated by the worms, no change of CYP expression and activity was found, except for a depression of CYP1A2 and 3A11 mRNAs in female mice.

Modulation of expression and activity of cytochrome P450s and alteration of praziquantel kinetics during murine schistosomiasis

In this study, we investigated the expression and activity of liver cytochrome P450s (CYPs) and praziquantel (PZQ) kinetics in mice infected with Schistosoma mansoni. Swiss Webster (SW) mice of both genders were infected (100 cercariae) on postnatal day 10 and killed on post-infection days (PIDs) 30 or 55. Non-infected mice of the same age and sex served as controls. Regardless of mouse sex, infection depressed the activities of CYP1A [ethoxy/methoxy-resorufin-O-dealkylases (EROD/MROD)], 2B9/10 [pentoxy/benzyloxy-resorufin-O-dealkylases (PROD, BROD)], 2E1 [p-nitrophenol-hydroxylase (PNPH)] and 3A11 [erythromycin N-demethylase (END)] on PID 55 but not on PID 30. On PID 55, infection decreased liver CYP mRNA levels (real-time reverse transcription-polymerase chain reaction). On PID 30, whereas mRNA levels remained unaltered in males, they were depressed in females. Plasma PZQ (200 and 400 mg/kg body weight intraperitoneally) levels were measured (high-performance liquid chromatography) at different post-treatment intervals. In males and females, infection delayed the PZQ clearance on PID 55, but not on PID 30. Therefore, it can be concluded that schistosomiasis down-modulated CYP expression and activity and delayed PZQ clearance on PID 55, when a great number of parasite eggs were lodged in the liver. On PID 30, when egg-laying was initiated by the worms, no change of CYP expression and activity was found, except for a depression of CYP1A2 and 3A11 mRNAs in female mice.

Up- and down-modulation of liver cytochrome P450 activities and associated events in two murine malaria models.

BACKGROUND: The mechanisms by which malaria up and down-regulates CYP activities are not understood yet. It is also unclear whether CYP activities are modulated during non-lethal malaria infections. This study was undertaken to evaluate the time course of CYP alterations in lethal (Plasmodium berghei ANKA) and non-lethal (Plasmodium chabaudi chabaudi) murine malaria. Additionally, hypotheses on the association of CYP depression with enhanced nitric oxide (NO) production, and of CYP2a5 induction with endoplasmic reticulum dysfunction, enhanced haem metabolism and oxidative stress were examined as well. METHODS: Female DBA-2 and C57BL/6 mice were infected with P.berghei ANKA or P. chabaudi and killed at different post-infection days. Infection was monitored by parasitaemia rates and clinical signs. NO levels were measured in the serum. Activities of CYP1a (ethoxyresorufin-O-deethylase), 2b (benzyloxyresorufin-O-debenzylase), 2a5 (coumarin-7-hydroxylase) and uridine-diphosphoglucuronyl-transferase (UGT) were determined in liver microsomes. Glutathione-S-transferase (GST) activity and concentrations of gluthatione (GSH) and thiobarbituric acid-reactive substances (TBARS) were determined in the liver. Levels of glucose-regulated protein 78 (GRP78) were evaluated by immunoblotting, while mRNAs of haemoxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) were determined by quantitative RT-PCR. RESULTS: Plasmodium berghei depressed CYP1a and 2b and induced 2a5 in DBA-2 mice. In P.berghei-infected C57BL/6 mice CYP activities remained unaltered. In both strains, GST and UGT were not affected by P.berghei. Plasmodium c. chabaudi depressed CYP1a and 2b and induced 2a5 activities on the day of peak parasitaemia or near this day. CYP2a5 induction was associated with over-expression of HO-1 and enhanced oxidative stress, but it was not associated with GRP78 induction, a marker of endoplasmic reticulum stress. Plasmodium chabaudi increased serum NO on days near the parasitaemia peak in both strains. Although not elevating serum NO, P.berghei enhanced iNOS mRNA expression in the liver. CONCLUSION: Down-regulation of CYP1a and 2b and induction of 2a5 occurred in lethal and non-lethal infections when parasitaemia rates were high. A contribution of NO for depression of CYP2b cannot be ruled out. Results were consistent with the view that CYP2a5 and HO-1 are concurrently up-regulated and suggested that CYP2a5 induction may occur in the absence of enhanced endoplasmic reticulum stress.

Distribution of ABCB1 polymorphisms among Brazilians: impact of population admixture.

INTRODUCTION: Interethnic admixture is a source of cryptic population structure that may lead to spurious genotype-phenotype associations in pharmacogenomic studies. We studied the impact of population stratification on the distribution of ABCB1 polymorphisms (1236C>T, 2677G>T/A and 3435C>T) among Brazilians, a highly admixed population with Amerindian, European and African ancestral roots. METHODS: Individual DNA from 320 healthy adults was genotyped with a panel of ancestry informative markers, and the proportions of African component of ancestry (ACA) were estimated. ABCB1 genotypes were determined by the single base extension/termination method. We describe the association between ABCB1 polymorphisms and ACA by fitting a linear proportional odds logistic regression model to the data. RESULTS: The distribution of the ABCB1 2677G>T/A and 3435C>T, but not the 1236C>T, SNPs displayed a significant trend for decreasing frequency of the T alleles and TT genotypes from White to Intermediate to Black individuals. The same trend was observed in the frequency of the T/nonG/T haplotype at the 1236, 2677 and 3435 loci. When the population sample was proportioned in quartiles, according to the individual ACA estimates, the frequency of the T allele and TT genotype at each locus declined progressively from the lowest (< 0.25 ACA) to the highest (> 0.75 ACA) quartile. Linear proportional odds logistic regression analysis confirmed that the odds of having the T allele at each locus decreases in a continuous manner with the increase of the ACA, throughout the ACA range (0.13-0.94) observed in the overall population sample. A significant association was also detected between the individual ACA estimates and the presence of the T/nonG/T haplotype in the overall population. CONCLUSION: Self-identification according to the racial/color categories proposed by the Brazilian Census is insufficient to properly control for population stratification in pharmacogenomic studies of ABCB1.

Função imediata em mandíbula: simplificação da técnica / Immediate function in mandible: technique simplification

A reabilitação da mandíbulas edêntulas por meio da submissão de implantes à função imediata é uma modalidade de tratamento com altos índices de sucesso que proporciona vantagens a pacientes e profissionais. Contudo, grande parte da população brasileira não tem acesso ao tratamento com oimplantes. Visando permitir que um maior número de pessoas tenha acesso à técnica foi desenvolvido um protocolo que reduz sensivelmente os custos e simplifica os procedimentos cirúrgicos e protéticos